Transport and localization of signaling proteins in ciliated cells

Abstract

Most cells in the human body elaborate cilia which serve a wide variety of functions, including cell and tissue differentiation during development, sensing physical and chemical properties of the extracellular milieu and mechanical force generation. Common among cilia is the transduction of external stimuli into signals that regulate the activities of the cilia and the cells that possess them. These functions require the transport and localization of specialized proteins to the cilium, a process that many recent studies have shown to be vital for normal cell function and, ultimately, the health of the organism. Here we discuss several mechanisms proposed for the transport and localization of soluble and peripheral membrane proteins to, or their exclusion from the ciliary compartment with a focus on how the structure of the cytoplasm and the size and shape of proteins influence these processes. Additionally, we examine the impact of cell and protein structure on our ability to accurately measure the relative concentrations of fluorescently tagged proteins amongst various cellular domains, which is integral to our understanding of the molecular mechanisms underlying protein localization and transport.

Figure 1. The Steric Volume Exclusion effect

Cell structures (A) and macromolecules (B) reduce the volume available to a solute molecule by an amount proportional to the radius of gyration (r_s) and the geometry of the space, represented in (A) as the distance between two parallel membranes (L). The result is that the total mass of a given protein found in a more structurally dense region of a cell will be lower than in a less dense region, and the relative magnitude of the difference will depend on the size of the molecule (C).

Figure 2. Cell structures and the resolution limit of fluorescence microscopy

Most cell structures are below the resolution limit of fluorescence microscopy, making quantitative comparisons of fluorescent protein concentrations amongst different cell regions challenging. This is particularly true of ciliated cells where the ~0.35 µm diameter of the cilium itself places it well below the resolution limit the excitation beam of confocal microscopy (the point spread function, psf, red ellipse) (A). Amphibian rod photoreceptors offer the advantage that both the ciliary compartment and the cell body are large enough to accommodate the full psf (B). In addition to the envelope geometry, sub-resolution structures within the cytoplasm, and the regions near them that are excluded to the radius of gyration of a given fluorescent molecule (shown in grey), occlude the sampling volume of the psf (C). Together these factors lead to variation in fluorescence levels measured between different cell regions despite uniform concentrations of fluorescent molecules within the contiguous aqueous cytoplasm. (See text for details)